title: Systematic Analysis Reveals Elongation Factor 2 and α-Enolase as Novel Interaction Partners of AKT2.
abstract: AKT2 is one of the three isoforms of the protein kinase AKT being involved in the modulation of cellular metabolism. Since protein-protein interactions are one possibility to convey specificity in signal transduction, we performed AKT2-protein interaction analysis to elucidate their relevance for AKT2-dependent cellular functions. We identified heat shock protein 90 kDa (HSP90), Cdc37, heat shock protein 70 kDa (HSP70), 78 kDa glucose regulated protein (GRP78), tubulin, GAPDH, α-enolase and elongation factor 2 (EF2) as AKT2-interacting proteins by a combination of tandem affinity purification and mass spectrometry in HEK293T cells. Quantitative MS-analysis using stable isotope labeling by amino acids in cell culture (SILAC) revealed that only HSP90 and Cdc37 interact stably with AKT2, whereas the other proteins interact with low affinity with AKT2. The interactions of AKT2 with α-enolase and EF2 were further analyzed in order to uncover the functional relevance of these newly discovered binding partners. Despite the interaction of AKT2 and α-enolase, which was additionally validated by proximity ligation assay (PLA), no significant impact of AKT on α-enolase activity was detected in activity measurements. AKT stimulation via insulin and/or inhibition with the ATP-competitive inhibitor CCT128930 did not alter enzymatic activity of α-enolase. Interestingly, the direct interaction of AKT2 and EF2 was found to be dynamically regulated in embryonic rat cardiomyocytes. Treatment with the PI3-kinase inhibitor LY294002 before stimulation with several hormones stabilized the complex, whereas stimulation alone led to complex dissociation which was analyzed in situ with PLA. Taken together, these findings point to new aspects of AKT2-mediated signal transduction in protein synthesis and glucose metabolism.
authors.affiliation: Department of Cardiovascular Physiology, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Bottermann
authors.forename: Michael
authors.initials: M
authors.lastname: Reinartz
authors.forename: Marian
authors.initials: M
authors.lastname: Barsoum
authors.forename: Sebastian
authors.initials: S
authors.lastname: Kötter
authors.forename: Axel
authors.initials: A
authors.lastname: Gödecke
mesh_terms: D002846:Chromatography, Affinity; D000091342:Endoplasmic Reticulum Chaperone BiP; D006019:Glycolysis; D057809:HEK293 Cells; D006801:Humans; D020652:Peptide Elongation Factor 2 / Q000378:metabolism*; D010751:Phosphopyruvate Hydratase / Q000378:metabolism*; D011485:Protein Binding; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
chemical_list: D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D020652:Peptide Elongation Factor 2; C494921:AKT2 protein, human; D051057:Proto-Oncogene Proteins c-akt; D010751:Phosphopyruvate Hydratase